When viewed with conventional incident light or transmitted light, non-absorbing microscopic specimens are not visible to the eye. The aforementioned apparatuses are used in order nevertheless to make structures of differing refractive index or differing thickness visible. Such structures of differing optical density that are not perceptible by the eye are generally referred to as phase specimens.
EP 069 263 A1, for example, describes one such phase contrast device. It substantially comprises an annular segment stop in the illumination unit that is imaged onto a phase ring in the objective. The outside diameter of the phase ring constitutes the diameter of the corresponding pupil. The transmission and phase of the rotationally symmetrical phase ring are predefined.
Contrast modulation microscopes based on Hoffman modulation contrast (HMC) are described in German Unexamined Applications DE 25 23 463 A1 and DE 25 23 464 A1. These possess a slit-shaped illumination stop, in the region of the condenser pupil, which is imaged onto a modulator in the objective exit pupil. The modulator comprises three strip-shaped zones of differing transmissivity that, in the embodiment corresponding to the existing art, exhibit no phase shift with respect to each other.
For simplification and in order to achieve a clearer presentation, the statements hereinafter will refer exclusively to the term “modulator.” This term encompasses on the one hand a modulator such as the one also described in the Unexamined Applications relating to HMC, and on the other hand to the phase ring such as the one known from the aforesaid EP 069 263 A1. Although the two components do not have identical functions, their effect is nevertheless comparable for purposes of the invention. In addition, in multifunctional microscopes they are already integrated into the same assembly. In such cases, the idea of the invention is of course also applicable to the entire assembly.
A substantial disadvantage of systems corresponding to the existing art is the fact that the phase shift between the zero-order diffraction and higher diffraction orders is almost impossible to adapt in specimen-specific fashion. DE 25 23 463 A1, for example, discloses an adaptation of phase by varying the choice of material for the modulator; but this as a rule requires modification of the microscope, and is very difficult to perform during normal utilization of the microscope. In addition, the fact that the materials of the modulator are predefined means that adaptation is possible only in predefined steps, and certainly not in continuous fashion. Acquisition of a large number of different modulators moreover entails considerable cost.
In order to achieve optimum contrast for a given geometry and transmissivity of the modulators, however, it is absolutely necessary for the phase also to be optimally adapted. When HMC is used, for example, to observe specimens that exhibit both large phase shifts and slight absorption, it is advantageous to introduce a phase shift in order to minimize halo effects. For very small phase specimens at the limits of detectability, it is furthermore advantageous to shift the phase through approximately 90 degrees in order to increase contrast.